Pax6a and Pax6b are required at different points in neuronal progenitor cell proliferation during...

Pax6a and Pax6b are required at different points in neuronalprogenitor cell proliferation during zebrafish photoreceptorregeneration

Ryan Thummel1, Jennifer M. Enright, Sean C. Kassen, Jacob E. Montgomery, Travis J.Bailey, and David R. Hyde*Department of Biological Sciences and the Center for Zebrafish Research University of NotreDame, Notre Dame, Indiana, 46556, USA

AbstractThe light-damaged zebrafish retina results in the death of photoreceptor cells and the subsequentregeneration of the missing rod and cone cells. Photoreceptor regeneration initiates withasymmetric Müller glial cell division to produce neuronal progenitor cells, which amplify, migrateto the outer nuclear layer (ONL), and differentiate into both classes of photoreceptor cells. In thisstudy, we examined the role of the Pax6 protein in regeneration. In zebrafish, there are two Pax6proteins, one encoded by the pax6a gene and the other encoded by the pax6b gene. Weintravitreally injected and electroporated morpholinos that were complementary to either thepax6a or pax6b mRNA to knockdown the translation of the corresponding protein. Loss of Pax6bexpression did not affect Müller glial cell division, but blocked the subsequent first cell division ofthe neuronal progenitors. In contrast, the paralogous Pax6a protein was required for later neuronalprogenitor cell divisions, which maximized the number of neuronal progenitors. Without neuronalprogenitor cell amplification, proliferation of resident ONL rod precursor cells, which can onlyregenerate rods, increased inversely proportional to the number of INL neuronal progenitor cells.This confirmed that Müller glial-derived neuronal progenitor cells are necessary to regeneratecones and that distinct mechanisms selectively regenerate rod and cone photoreceptors. This workalso defines distinct roles for Pax6a and Pax6b in regulating neuronal progenitor cell proliferationin the adult zebrafish retina and increases our understanding of the molecular pathways requiredfor photoreceptor cell regeneration.

KeywordsPax6; morpholino; Müller glia; neuronal progenitor; retina; cell migration; stem cell

© 2009 Elsevier Ltd. All rights reserved.*Correspondence should be addressed to: David R. Hyde, Ph.D., Department of Biological Sciences and the Center for ZebrafishResearch, University of Notre Dame, Notre Dame, Indiana, 46556, USA, Phone: 1-(574) 631-8054, Fax: 1-(574) 631-7413,[email protected] address: Department of Anatomy and Cell Biology and Department of Ophthalmology Wayne State University School ofMedicine, Detroit, MI, 48201, USAPublisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ourcustomers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review ofthe resulting proof before it is published in its final citable form. Please note that during the production process errors may bediscovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptExp Eye Res. Author manuscript; available in PMC 2011 May 1.

Published in final edited form as:Exp Eye Res. 2010 May ; 90(5): 572–582. doi:10.1016/j.exer.2010.02.001.

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INTRODUCTIONThe zebrafish retina undergoes persistent neurogenesis throughout its life (Marcus et al.,1999; Otteson et al., 2001; Otteson and Hitchcock, 2003; Raymond et al., 2006). All retinalneurons, except rods, are derived from a population of neuronal stem cells located at thecircumferential marginal zone (Marcus et al., 1999; Raymond et al., 2006). Rods aregenerated from a limited number of Müller glial cells that divide asymmetrically to producea neuronal progenitor cell that migrates to the ONL (Bernardos et al., 2007; Raymond et al.,2006). Upon reaching the ONL, this cell is called a rod precursor cell because it iscommitted to differentiating into a rod photoreceptor (Hitchcock and Raymond, 2004;Morris et al., 2008a; Otteson et al., 2001; Raymond et al., 2006). These rod precursor cellscontinue to proliferate at a low rate until they differentiate into rods. While a variety ofgenes are differentially expressed in the neuronal progenitor and rod precursor cells(Hitchcock and Raymond, 2004; Morris et al., 2008b; Otteson et al., 2001; Raymond et al.,2006; Stenkamp, 2007), it is the spatial location that most accurately defines anddistinguishes these two cell types at the present time.

A variety of insults to the zebrafish retina induces neuronal death, followed by a significantincrease in the number of proliferating Müller glia to produce neuronal progenitors thatcontinue to undergo cell division and accurately differentiate into the lost neurons(Bernardos et al., 2007; Braisted et al., 1994; Cameron, 2000; Fausett and Goldman, 2006;Fimbel et al., 2007; Hitchcock et al., 1992; Maier and Wolburg, 1979; Raymond et al., 2006;Vihtelic and Hyde, 2000; Wu et al., 2001; Yurco and Cameron, 2005). While thisregeneration response is largely absent in mammalian retinas (Bringmann et al., 2006), anumber of reports indicate that mammalian Müller glia possess features that are similar toadult neuronal stem cells (Lawrence et al., 2007; Rowan and Cepko, 2004). Elucidating themolecules that regulate these proliferative events in a robustly regenerating species couldlead to therapies for retinal degenerative diseases.

Constant intense light treatment of dark-adapted albino zebrafish selectively kills rod andcone photoreceptors (Vihtelic and Hyde, 2000), which induces approximately 50% of theMüller glia to divide and produce neuronal progenitor cells (Thummel et al., 2008b). Theseneuronal progenitors initiate expression of stem/progenitor cell markers, including Pax6,Rx1, and Chx10 (Raymond et al., 2006; Thummel et al., 2008a), continue to proliferate, andmigrate from the inner nuclear layer (INL) to the outer nuclear layer (ONL). There theyultimately differentiate into rods and cones to replace the lost photoreceptors (Vihtelic andHyde, 2000). While Ascl1a and Stat3 have been shown to be required for Müller glialproliferation (Fausett et al., 2008; Kassen et al., 2010), there are currently no knownmolecules that specifically regulate the subsequent proliferation of the neuronal progenitorcells.

Pax6 plays a pivotal role in eye development in species as diverse as flies, zebrafish, mice,and humans (Nornes et al., 1998; Xu et al., 2007). Pax6 is also expressed in the neuronalprogenitor cells during regeneration of the adult zebrafish retina (Raymond et al., 2006;Thummel et al., 2008a). Zebrafish possess two paralogous pax6 genes (pax6a and pax6b),which encode functionally redundant proteins that regulate formation and differentiation ofthe retina and lens (Blader et al., 2004). However, neither the redundancy nor the function ofthe Pax6 paralogs has been investigated in the regenerating retina.

We demonstrated that conditional knockdown of either Pax6a or Pax6b protein expressiondisrupted discrete events during retinal regeneration. Knockdown of Pax6b expressioninhibited the subsequent division of the Müller glia-derived INL neuronal progenitor. Incontrast, reduction of Pax6a expression allowed this initial neuronal progenitor cell division,

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but it prevented later cell divisions. The reduced number of INL neuronal progenitor cells inboth Pax6 knockdown retinas resulted in a corresponding loss of regenerated cones, but notrods. This indicated that Müller glia-derived INL progenitors were required for cone cellregeneration, while rods regenerated from increased amplification of resident ONL rodprecursors. This work describes a novel Pax6-dependent mechanism to regulate the extent ofneuronal progenitor cell amplification.

MATERIALS AND METHODSFish maintenance

Wild-type AB and albino strains of zebrafish (Danio rerio) were maintained in the Centerfor Zebrafish Research at the University of Notre Dame Freimann Life Science Center. Theadults used for these studies were 6 to 12 months old and 4 to 5 cm in length. Fish were fedbrine shrimp and flake food three times daily and maintained under a daily light cycle of 14hours light (250 lux):10 hours dark at 28.5°C (Detrich et al., 1999; Westerfield, 1995). Allprotocols used in this study were approved by the animal use committee at the University ofNotre Dame and are in compliance with the ARVO statement for the use of animals invision research.

MorpholinosThis study utilized five lissamine-tagged morpholinos (Gene Tools LLC, Philomath, OR).The Gene Tools Standard Control morpholino ( 5’ –CCTCTTACCTCAGTTACAATTTATA – 3’) was used as a non-specific control, because ithas no complementary sequence in the zebrafish genome. Two different anti-pax6morpholinos were used that were based on the previously published anti-pax6a and anti-pax6b morpholino sequences (Blader et al., 2004). The anti-pax6a morpholino iscomplementary to the 5' UTR of only the pax6a mRNA (5’ –TTTGTATCCTCGCTGAAGTTCTTCG – 3’) and the anti-pax6b morpholino iscomplementary to the 5' UTR of only the pax6b mRNA (5’ –CTGAGCCCTTCCGAGCAAAACAGTG – 3’). Two 5-base mismatch control morpholinoswere derived from the anti-pax6a and anti-pax6b sequences, 5mis pax6a MO-1 (5’ –TTTCTATGCTCGCTCAACTTGTTCG-3’) and 5mis pax6b MO-2 (5’ –CTCAGACCTTCCGAGCCAAATACTG – 3’), respectively, with the mismatched basesunderlined. Morpholinos were resuspended in water to a working concentration of 3 mM.

Immunoblot of Pax6 expression in morphant zebrafish embryosMorpholinos were micro-injected into 1–4 cell stage embryos and protein was isolated forimmunoblot analysis as previously described (Thummel et al., 2008b). Embryos wereinjected with either the anti-pax6a morpholino, the anti-pax6b morpholino, or a cocktail ofboth morpholinos. Total protein was isolated from uninjected and morphant embryos at 48hours post fertilization (hpf). Total protein equivalent of 1 embryo was combined with 4xsample buffer and 10x reducing buffer (Invitrogen, Carlsbad, CA), incubated at 70°C for 10min, and loaded onto a 4–12% SDS-PAGE (Invitrogen). Following electrophoresis, thesample was transferred to a PVDF membrane (Amersham, Pittsburgh, PA). The membranewas blocked in PBS/5% nonfat dry milk/0.1% Tween-20 and then incubated with a rabbitpolyclonal anti-Pax6 antiserum (diluted 1:5000, Covance, Berkeley, CA) overnight at 4°C inblocking buffer. The anti-Pax6 antiserum recognizes both zebrafish Pax6 proteins. Themembrane was washed in PBS/0.1% Tween-20, and then incubated with an anti-rabbitHRP-conjugated secondary antibody (diluted 1:10,000, Amersham). The ECL-Plus system(Amersham) was used to detect the secondary antibody as previously described (Thummelet al., 2008b; Vihtelic et al., 1999).

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Constant intense-light treatmentRod and cone photoreceptors were damaged in albino zebrafish using constant intense-lighttreatment (Vihtelic and Hyde, 2000; Vihtelic et al., 2006). Fish were dark adapted for 14days and then were placed in tanks between four 250W halogen bulbs, which generated alight intensity of 6,000 lux. This constant intense light was maintained for up to 4 days, afterwhich the fish were returned to standard light conditions (250 lux, 14 hours light: 10 hoursdark).

Injection and electroporation of morpholinos in adult zebrafish retinasMorpholino injection and electroporation was performed as previously describedimmediately prior to light treatment (Thummel et al., 2008b). Briefly, dark-adapted adultalbino zebrafish were anesthetized, the outer most component of the cornea was removedwith small forceps, a small incision was made in the cornea, and 0.5 µl of a 3 mMmorpholino solution was injected into the vitreous of the left eye using a Hamilton syringe.The 3 mM cocktail that contained both anti-pax6a and anti-pax6b morpholinos had a finalconcentration of 1.5 mM of each morpholino. Immediately following the injections, the lefteye was electroporated with a CUY21 Square Wave Electroporator (Protech International,Inc., San Antonio, TX), using 2 consecutive 50-msec pulses at 75 V with a 1 sec pausebetween pulses. A 3 mm diameter platinum plate electrode (CUY 650-P3 Tweezers, ProtechInternational, Inc.) was used to drive the morpholino into the central and dorsal regions ofthe retina.

Bromodeoxyuridine labeling in vivoDark-adapted adult albino zebrafish were either uninjected or injected and electroporatedwith a 5-base mismatch control morpholino, anti-pax6a morpholino, or anti-pax6bmorpholino as described above. After 24 hours of light treatment, fish were placed in a tankcontaining system water and 5 mM bromodeoxyuridine (BrdU) for 48 hours, as previouslydescribed (Bernardos et al., 2007).

ImmunohistochemistryFish were euthanized at various time points during and following the constant lighttreatment by anesthetic overdose in 2-phenoxyethanol and eyes were collected. Unlessotherwise noted, eyes were fixed in 9:1 ethanolic formaldehyde (100% ethanol:37%formaldehyde). After incubating overnight in fixative at 4°C, eyes were washed in 5%sucrose/1X PBS and cryoprotected in 30% sucrose/1X PBS overnight at 4°C. Eyes weresubsequently washed in 1:1 Tissue Freezing Medium (TFM, Triangle Biomedical Sciences,Durham, NC):30% sucrose/1X PBS at 4°C overnight. The retinas were embedded in TFMand cryosectioned at a thickness of 14 microns. Frozen sections were dried at 50°C for twohours and subsequently stored at −80°C.

Prior to immunolabeling, slides were thawed for 20 minutes at 50°C and then rehydrated in1X PBS. Retinal sections were incubated in blocking solution 1X PBS/2% NGS (normalgoat serum)/0.2% Triton X-100/1% DMSO for 1 hour at room temperature prior toincubating overnight at 4°C with the primary antibody diluted in blocking buffer. Primaryantibodies used for these studies include the mouse anti-PCNA monoclonal antibody(1:1000, clone PC10, Sigma Chemical, St. Louis, MO), rabbit anti-PCNA polyclonalantiserum (1:1500, Abcam, Cambridge, MA), rat anti-BrdU polyclonal antiserum (1:200,Accurate Chemical, Westbury, NY) rabbit anti-rhodopsin polyclonal antiserum (1:5000,(Vihtelic et al., 1999)), rabbit anti-UV opsin polyclonal antiserum (1:1000, (Vihtelic et al.,1999)), rabbit anti-blue opsin polyclonal antiserum (1:250, (Vihtelic et al., 1999)), rabbitanti-Pax6 polyclonal antiserum (1:100, Covance), rabbit anti-GFP polyclonal antiserum (1:

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500, Abcam), and mouse anti-glutamine synthetase monoclonal antibody (1:500, ChemiconInternational, Temecula, CA). A modified antigen retrieval protocol was utilized for Pax6immunostaining as previously described (Thummel et al., 2008a). Following incubation inprimary antibody solution, sections were washed in 1X PBS/0.05% Tween-20 and thenincubated for one hour at room temperature with the secondary antibody diluted 1:500 in 1XPBS/0.05% Tween-20. Secondary antibodies included Alexa Fluor goat anti-primary IgG488, 568, 594, and 647 (Molecular Probes, Eugene, OR). Nuclei were labeled with TO-PRO-3 (Molecular Probes) at a 1:750 dilution in 1X PBS/0.05% Tween-20. Sections werewashed again in 1X PBS/0.05% Tween-20 and 1X PBS before mounting with glasscoverslips and ProLong Gold (Molecular Probes).

For BrdU immunolabeling, eyes were fixed in a 4% paraformaldehyde solution containing5% sucrose/1X PBS, and embedded and sectioned as described above. Followingrehydration, sections were washed in 1X PBS/0.3% Triton X-100 for 5 minutes, washed twotimes (5 minutes each) in DNaseI buffer (4.2 mM MgCl2/150 mM NaCl/0.3% TritonX-100/0.5% DMSO), and then treated with 50 Kunitz/mL DNaseI (diluted in DNaseIbuffer) for 10 minutes at room temperature. Sections were then washed twice in 1X PBS/0.3% Triton X-100 for 5 minutes at room temperature. Sections were blocked, incubatedwith anti-BrdU primary antiserum, washed and incubated with secondary antibody asdescribed above.

For BrdU and PCNA colabeling, the BrdU protocol was followed and then sections wereplaced in 10 mM sodium citrate (pH 6.0) at 95° C for 20 minutes. Sections were left to coolfor 20 minutes, washed twice in 1X PBS/0.3% Triton X-100 for 5 minutes, and then blockedas described above. BrdU and PCNA immunolocalizations were then performed asdescribed above.

For TUNEL and PCNA colabeling, dark-adapted adult albino zebrafish were eitheruninjected or injected and electroporated with a 5-base mismatch control morpholino, anti-pax6a morpholino, or anti-pax6b morpholino. After 72 hours of constant light treatment,eyes were fixed, infiltrated and sectioned as described above. Following a wash in 1X PBS,the tissue was permeablized with a 5 minute wash in ice cold 1X PBS/ 0.1% NaCitrate/0.1% TritonX-100, followed by a wash in room temperature 1X PBS. TdT labeling(Clontech; Mountain View, CA) was performed per manufacturer’s instructions, with oneexception: biotin-conjugated dNTPs (New England Biolabs; Ipswich, MA) were used inplace of the FITC-conjugated dNTPs. The labeling reaction was stopped by adding 2X SSCand the tissue was washed twice in 1X PBS. Blocking and primary antibody incubation wereperformed as described above. A fluorescent StrepAvidin-conjugate (1:200, MolecularProbes) was added with the secondary antibody mix (described above) and the slides wereplace in the dark for 1 hour at room temperature, washed in 1X PBS, and mounted withglass coverslips.

Statistical analysis of confocal microscopyConfocal microscopy was performed with either a 1024 BioRad or Leica TCS SP5 confocalmicroscope. To maintain consistency between eyes and experimental groups, only retinalsections that contained or were immediately adjacent to the optic nerve were utilized.Quantification of individual cell types or nuclei was obtained from a linear distance of 740microns in the central dorsal retina, which excluded the margin and optic nerve andcorresponded to approximately half the linear distance of the dorsal retina. All quantificationdata is presented in Table 1. As clearly noted in the text and Table 1, between 6 and 15retinas were analyzed per experiment, which is a standard sample size for adult zebrafishstudies.

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In each experiment, all data sets were first analyzed with a repeated measures ANOVA tofirst assess whether there were any significant differences between the various groups. Thep-value for each of these tests were reported in Table 1. In experiments where the ANOVArevealed a statistically significant difference (p<0.001), two-sample t-tests were performedon each pair of samples and Bonferroni-corrected to account for the number of multipletests. The individual significance levels (p-values) are reported in the body of the text.

RESULTSPax6a and Pax6b expression is efficiently reduced by electroporated morpholinos

As we previously reported, Pax6 was constitutively expressed in the amacrine cells in theINL and ganglion cells in the GCL in the undamaged adult zebrafish retina (Fig. 1A;(Thummel et al., 2008a)). Constant intense light treatment of dark-adapted albino zebrafishresulted in photoreceptor cell death and increased proliferation of rod precursor cells in theONL at 16 hours (Fig. 1B), followed by Müller glia proliferation at 36 hours (Fig. 1C). Pax6expression began colabeling with the proliferative marker PCNA in columns of neuronalprogenitors after 36 hours of constant light and persisted through 96 hours (Fig. 1D, E), asthey proliferated and migrated towards the ONL. Pax6 expression in the neuronalprogenitors was largely absent by 3 days post light treatment (Thummel et al., 2008a), whenthe neuronal progenitors had finished migrating to the ONL and were differentiating intorods and cones. This temporal and spatial expression pattern of Pax6 corresponded to whenneuronal progenitor cell proliferation and migration would be regulated.

We confirmed the efficiency and specificity of two previously published anti-pax6morpholino sequences to knockdown Pax6a and Pax6b protein expression (Blader et al.,2004) by microinjecting the morpholinos into 1–4 cell stage embryos (see Methods).Subsequent immunoblot analysis showed that each anti-pax6 morpholino knocked down thedesired Pax6 protein (Fig. 2).

We injected and electroporated either the anti-pax6 or 5-base mismatch control (5mis)morpholinos into dark-adapted albino zebrafish retinas immediately before beginning theconstant intense light treatment. After 72 hours in constant light, retinas wereimmunolabeled for Pax6 expression. Uninjected control retinas (Fig. 1F) exhibited Pax6expression in the ganglion cells, amacrine cells, and neuronal progenitor cell clusters (Fig.1F). Retinas injected and electroporated with either the 5mis MO-1 or 5mis MO-2morpholinos were indistinguishable from the uninjected control, except for slightly elevatedPax6 expression in the inner plexiform layer (Fig. 1G, H). The pax6a morphant and pax6a/pax6b double morphant both possessed significantly reduced Pax6 expression in theganglion cells, amacrine cells, and neuronal progenitor cell clusters relative to the controls(Fig. 1I, K). In contrast, the pax6b morphant exhibited normal Pax6 expression in theganglion and amacrine cells, but reduced expression in the neuronal progenitor cell clustersrelative to the controls (Fig. 1J). Thus, the ganglion and amacrine cells primarily expressedthe Pax6a paralog, while the neuronal progenitors appeared to express both paralogs.

Pax6a and Pax6b are required at different points during neuronal progenitor proliferationTo examine if knockdown of either Pax6a or Pax6b expression affected cell proliferation inthe regenerating retina, dark-adapted albino zebrafish were injected and electroporated withexperimental and control morpholinos and then placed in constant light. After 36 hours ofconstant light, retinas in all groups exhibited PCNA-positive rod precursor cells and Müllerglia (Supplementary Fig. 1A–D). Thus, neither Pax6 protein appeared to be required foreither Müller glial or rod precursor cell division.

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To examine if Pax6 was necessary for neuronal progenitor cell division, we examined themorphant retinas after 72 hours of constant light. The control retinas each possessed clustersof PCNA-positive neuronal progenitor cells associated with Müller glial cell processes (Fig.3A–C). The pax6a morphant retina also exhibited clusters of PCNA-positive neuronalprogenitors (Fig.3D), however, there was an average of only 3.8 ± 0.1 PCNA-positiveprogenitors in each pax6a morphant retinal cluster relative to 7.9 ± 0.7 PCNA-positiveprogenitors in each control cluster (Table 1; compared to uninjected control, p=0.003). Boththe pax6b morphant and pax6a/pax6b double morphant contained an average of only 2PCNA-positive neuronal progenitors per cluster (Fig. 3E, F; Table 1; compared touninjected control, p=0.0005 and p=0.0007, respectively). Thus, knockdown of Pax6bexpression inhibited the subsequent division of the Müller glia-derived INL neuronalprogenitor. In contrast, reduction of Pax6a expression allowed this initial neuronalprogenitor cell division, but it prevented later cell divisions.

To independently confirm the PCNA immunolabeling, we labeled proliferating progenitorswith BrdU between 24 and 72 hours of light treatment. As expected, BrdU colabeled withPCNA in the dividing neuronal progenitors after 72 hours of constant light treatment (Fig.4A). Furthermore, the BrdU-labeled neuronal progenitors were associated with GFAP-positive Müller glial processes (Fig. 4B). Three days after completing the four days ofconstant intense light treatment, large numbers of INL neuronal progenitor cells hadmigrated to the ONL (Raymond et al., 2006) and were observed to be BrdU-positive (Fig.4C). In addition, the Müller glial cell nuclei that initially divided around 36 hours ofconstant light treatment remained BrdU-positive (Fig. 4C).

After 72 hours of constant light treatment, control retinas incorporated BrdU in clusters ofneuronal progenitors that each contained an average of 7.4 to 8.1 nuclei (Fig. 4D–F; Table1). In contrast, the pax6a morphant retinas (Fig. 4G) contained an average of only 4.6 BrdU-labeled nuclei per cluster (Table 1; compared to uninjected control, p=0.01). Further, thepax6b morphant and pax6a/pax6b double morphant retinas possessed an average of only 2.3and 2.0 BrdU-labeled nuclei per cluster, respectively (Fig. 4H, I; Table 1, compared touninjected control, p=0.001 and p=0.0008, respectively). These data suggest that theasymmetric Müller glial cell division to produce the first neuronal progenitor does notrequire Pax6 expression, while the subsequent division of the neuronal progenitor cell needsPax6b expression. Furthermore, Pax6a was only required for one of the final rounds ofprogenitor cell division in each cluster.

Reduced numbers of neuronal progenitors observed in pax6 morphants is not a result ofincreased cell death

We previously reported that knockdown of PCNA during constant intense light treatmentinhibited Müller glial cell division (Thummel et al., 2008b). Because Müller glial celldivision cannot occur without necessary levels of PCNA, these activated Müller glia died,with TUNEL-positive Müller glia observed 12 hours following the failed division (Thummelet al., 2008b). Given that we observed reduced numbers of dividing neuronal progenitors inpax6a and pax6b morphant retinas, it is possible that Pax6 is required for neuronalprogenitor cell survival. We took two approaches to determine whether this was the case.First, control and morphant retinas were analyzed for the presence of TUNEL-positivenuclei at 72 hours of constant light treatment. Based on our previous findings, if neuronalprogenitors were dying as a result of Pax6 knockdown, then TUNEL-positive nuclei wouldbe present in the INL at this time point. Similar to a previous report (Bernardos 2007), veryfew TUNEL-positive nuclei were observed in the ONL of either the control or morphantretinas (Fig 5). Importantly, no TUNEL-positive nuclei were visualized in the INL of thecontrols, pax6a or pax6b morphant retinas (Fig. 5). Colabeling with PCNA confirmed thatneuronal progenitors were present in these retinas, but were not TUNEL-positive (Fig. 5).

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Second, we analyzed control and morphant retinas after 4 days of constant intense lightfollowed by recovery in 3 days of standard light. We found the PCNA-positive INL nucleipersisted in both the pax6b and pax6a/pax6b morphants (Fig. 6K, L), indicating that thesecells have not died, but were still present many days after their initial entrance into the cellcycle. Together, these data show that neuronal progenitors are not dying as a result of eitherPax6a or Pax6b knockdown.

Rod precursor proliferation is inversely proportional to the number of INL neuronalprogenitors

To examine the subsequent effects of Pax6 knockdown, we injected and electroporated themorpholinos into dark-adapted albino zebrafish retinas and analyzed the retinas after 4 daysof constant intense light followed by recovery in 3 days of standard light. The control retinaspossessed newly regenerated rhodopsin-positive rod photoreceptors (Fig. 6A–C), withPCNA expression primarily restricted to the ONL rod precursor cells (Fig. 6G–I). The pax6amorphant retina exhibited reduced rhodopsin expression relative to the control retinas (Fig.6D), suggesting that fewer rods had regenerated in the pax6a morphant compared to thecontrol retinas. Strikingly, both the pax6b morphant and pax6a/pax6b double morphantretinas possessed only isolated and short rhodopsin-positive rod outer segments (Fig. 6E, F),suggesting that even fewer rods had regenerated relative to the pax6a morphant.

We also quantified the number of PCNA-positive nuclei at this timepoint. As noted above,the pax6b morphant and pax6a/pax6b double morphant retinas still possessed the doubletsof PCNA-positive INL cells (Fig. 6K, L), which were first observed at 72 hours of constantlight (Fig. 3 and Fig. 4). Thus, in the absence of Pax6b, INL neuronal progenitors failed tomigrate to the ONL. However, surprisingly, the pax6b morphant and pax6a/pax6b doublemorphant retinas contained over twice the number of PCNA-positive ONL cells relative tothe controls (Table 1; compared to uninjected control, p=0.0008 and p=0.001, respectively).Given the apparent lack of neuronal progenitor cell migration from the INL in thesemorphant retinas, the large number of proliferating ONL cells represented resident ONL rodprecursors. Therefore, these data suggested that the number of INL neuronal progenitors thatmigrated to the ONL proportionally reduced the number of proliferating ONL rod precursorcells. Data from the pax6a morphants further supported this hypothesis. Similar to thecontrol retinas, the pax6a morphant retinas contained very few PCNA-positive neuronalprogenitors in the INL at this time point (Fig. 6G–J), suggesting that the few neuronalprogenitors observed in the pax6a morphant retinas did migrate to the ONL. In addition, thepax6a morphant retina contained an intermediate number of PCNA-positive ONL nuclei(118.4 ± 20.3), which was greater than the control retinas (70.5 to 78.4, Table 1; p=0.04),but fewer than observed in the pax6b retinas (161 ± 11.4, Table 1). Together, these datasuggest that rod precursor proliferation is inversely proportional to the number of INLneuronal progenitors that migrate to the ONL during photoreceptor regeneration.

Pax6-stimulated neuronal progenitors are required for cone cell regenerationThe failure of neuronal progenitors to migrate to the ONL in the pax6b morphant and pax6a/pax6b double morphant retinas allowed us to determine if they were required to regenerate aspecific class of photoreceptor. At 28 days following the completion of the constant lighttreatment, the control and experimental retinas all exhibited robust rhodopsin localization(Fig. 7A–E) and an equivalent number of rod photoreceptor nuclei in the ONL (Table 1). Incontrast, significantly fewer long and short single cones were present in the pax6b morphantand pax6a/pax6b double morphant retinas relative to the controls (Fig. 7I, J, N and O; Table1; compared to uninjected control, p=0.002 and p=0.002 for long single cones, respectively).Furthermore, the pax6a morphant retina exhibited a level of cone cell regenerationintermediate to the controls and the pax6b morphant and pax6a/pax6b double morphants

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(Fig. 7H, M; Table 1; compared to uninjected control, p=0.04 for long single cones),although this value was not statistically significant relative to the controls. This level of conecell regeneration corresponded to the relative number of neuronal progenitors present ineach INL cluster (Fig. 3 and Fig. 4; Table 1). This suggested that Pax6-dependent INLneuronal progenitor proliferation was necessary for regeneration of cones and not rods,while the ONL rod precursors were sufficient for regeneration of rods.

DISCUSSIONUnlike the mammalian retina, the adult zebrafish possess the ability to specificallyregenerate all retinal cell types (Bernardos et al., 2007; Fausett and Goldman, 2006; Fimbelet al., 2007; Raymond et al., 2006; Vihtelic and Hyde, 2000; Wolburg, 1975; Yurco andCameron, 2005). Zebrafish retinal regeneration originates from two stem cell populations:the INL Müller glia and the ONL rod precursors (Bernardos et al., 2007; Fausett andGoldman, 2006; Morris et al., 2008b; Morris et al., 2005; Thummel et al., 2008b; Yurco andCameron, 2005). Müller glia reenter the cell cycle and produce multipotent neuronalprogenitors, which continue to divide and migrate to the damaged nuclear layer, where theydifferentiate into the neuronal cell types that were lost. Rod precursors can also reenter thecell cycle when rod photoreceptors are lost and differentiate into only rods (data not shown,Fig. 3 and Fig. 4). In the case of chronic rod cell death (Morris et al., 2005), however, onlyrod precursor proliferation is observed.

This study builds on the observed increase in pax6b mRNA expression and the increasedPax6 protein expression during regeneration of the light-damaged retina (Raymond et al.,2006; Thummel et al., 2008a). We also previously demonstrated that the pax6b mRNAexpression increased first, immediately following Müller glial cell division, followed byincreased pax6a mRNA expression when neuronal progenitors were undergoing rapid celldivisions (Thummel et al., 2008a). Using morpholinos that were electroporated into theretina to knockdown the expression of either one or both of the Pax6 proteins, we examinedthe functions of Pax6a and Pax6b during regeneration of rods and cones. Knockdown ofPax6a, Pax6b, or both proteins had no effect on the ability of Müller glia or rod precursors toenter the cell cycle (Supplementary Fig. 3). Because we never observed Pax6 expression inrod precursors, this was not surprising. However, it was reported that very weak Pax6expression was detected in the Müller glia of undamaged retinas (Bernardos et al., 2007).Although we failed to detect Pax6 expression in the Müller glia prior to retinal damage (Fig.1A), our functional data clearly demonstrated that neither Pax6 paralog was required for theinitiation of Müller glial cell division. Furthermore, these data showed that the previously-published morpholinos used in this study did not cause any non-specific delays in theregenerative timecourse (Blader et al., 2004).

The adult zebrafish regeneration response initiates with the Müller glia reentering the cellcycle (Fig. 8). This could be either an asymmetric cell division to yield a Müller glial celland a neuronal progenitor cell or the Müller glial cell may dedifferentiate and then divide toyield two neuronal progenitor cells. Regardless of the mechanism underlying this first celldivision, the second cell division requires the Pax6b protein. In contrast, knockdown of thePax6a protein does not disrupt this second cell division, which may be because the pax6agene is not expressed at this point in regeneration. This temporal sequence is consistent withthe expression of pax6b increasing prior to pax6a during regeneration of the light-damagedretina, as we previously reported (Thummel et al., 2008a). Alternatively, it is possible thatPax6b is a stronger regulator of neuronal progenitor proliferation, as has been previouslysuggested (Nornes et al., 1998). Thus, we cannot differentiate if the Pax6b protein regulatesa different set of genes than Pax6a, which separates the functions of these two proteins atthis second cell division, or if the two proteins regulate the same set of target genes and that

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they differ simply in their temporal expression and/or strength of activation. Furthermore, itis unclear if photoreceptor cell regeneration requires Pax6 expression in the neuronalprogenitor cells or in the amacrine and ganglion cells. While it seems most likely that Pax6expression in these neuronal progenitor cells would be required to regulate theirproliferation, it is possible that Pax6 expression in the adjacent amacrine and ganglion cellsmay be necessary to regulate neuronal progenitor cell proliferation through a secreted factor.Definitive proof of either of these two models will require the knockdown of Pax6expression in specific cell types.

The neuronal progenitors continue to divide, if Pax6b is present, until they reach 4 cells ineach INL cluster (Fig. 8). At this point, the Pax6a protein is required to activate theexpression of specific target genes to allow the continued proliferation of the progenitors.However, in the absence of Pax6a, the four progenitors will migrate to the ONL sometimebetween 3 and 7 days of starting the constant light treatment. In contrast, the two progenitorcell clusters in the pax6b morphant are still present 7 days after starting the constant lighttreatment. The persistence of these progenitors and the absence of TUNEL-positive nuclei inthe INL (Fig. 5) clearly show that neuronal progenitors are not dying as a result of Pax6bknockdown. However, it is unclear what mechanism allows for the migration of the fourneuronal progenitors in the pax6a morphant retinas, but does not allow for the migration ofthe two neuronal progenitors in the pax6b and pax6a/pax6b morphant retinas. Because BrdUwould incorporate into both the INL and ONL progenitors at all time points, a standard celltracking study would not differentiate the origin of the ONL progenitors. Thus, we cannotdifferentiate between Müller glial-derived rod photoreceptors and rod precursor-derived rodphotoreceptors during the regeneration response. However, the development of new genetictechnologies, transgenic zebrafish lines, and antisera that are specific to the individual celllineages may allow for future resolution of this question (Bernardos and Raymond,2006;Hitchcock and Kakuk-Atkins, 2004;Kassen et al., 2007;Morris et al., 2008b).

The failure of the neuronal progenitors to migrate to the ONL in the pax6b morphant andpax6a/pax6b double morphant resulted in twice the number of proliferating ONL rodprecursors as the control retinas (Table 1). In contrast, the pax6a morphant retina producedclusters consisting of four proliferating neuronal progenitor cells that appeared to migrate tothe ONL, which was approximately half the number as the control retinas. This resulted inan intermediate number of proliferating ONL rod precursors relative to the control and thepax6b morphant retinas. Taken together, the proliferation of the resident rod precursor cellsin the ONL, which are committed to becoming rods, may be negatively regulated by therelative number of INL neuronal progenitors that migrate to the ONL during regeneration ofthe light-damaged retina. This model implies that the migrating neuronal progenitors fromthe INL repress the proliferation of the resident ONL rod precursor cells. Furthermore, theincreased number of PCNA-positive rod precursor cells in both the pax6b and the pax6amorphant retinas clearly shows that rod precursor proliferation is not directly dependent ofPax6. It is likely that many of the increased number of proliferating ONL rod precursors inthe pax6b and pax6a morphant retinas, relative to the control retinas, ultimately die becauseall the retinas contain nearly equivalent numbers of regenerated rods. However, we wereunable to detect the death of these proliferating rod precursor cells in the pax6a and pax6bmorphant retinas, likely due to our inability to identify the proper time point when this celldeath was occurring.

The control and pax6 morphant retinas all exhibited an equivalent number of regeneratedrods. The apparent failure of the pax6b morphant and pax6a/pax6b double morphant retinasto generate neuronal progenitors that migrated to the ONL indicates that the regeneratedrods in these morphants were derived entirely from rod precursors. The regeneration of rodsfrom resident ONL rod precursors in the light-damaged pax6b morphant retina is consistent

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with the persistent proliferation and attempted regeneration of rods in the chromic rod-lesstransgenic line Tg(XOPS::mCFP)QO1 (Morris et al., 2008b; Morris et al., 2005).Furthermore, examination of the number of regenerated cones in the control and pax6morphant retinas was proportional to the number of neuronal progenitors that migrated tothe ONL. Thus, this is the first report to demonstrate that the regeneration of cones requiresproliferation of the Müller glial-derived neuronal progenitor cells, while rods can beregenerated from increased amplification of resident ONL rod precursors.

In addition to adding to the growing number of works that seek to characterize the molecularrequirements of Müller glia-derived neuronal regeneration, our results detail three novelfindings. First, Pax6a and Pax6b are the first transcription factors shown to specifically anddifferentially regulate neuronal progenitor cell proliferation during regeneration of the adultvertebrate retina. This is in stark contrast to the requirement of Ascl1a and Stat3 for theinitial proliferation of the Müller glia in the damaged zebrafish retina (Fausett et al., 2008;Kassen et al., 2010). Thus, Pax6a and Pax6b define steps subsequent to Ascl1a and Stat3 inretinal regeneration. Second, our results demonstrate an inverse relationship between thenumber of Müller glial-derived INL neuronal progenitors migrating to the ONL and theextent of proliferation of the resident ONL rod precursors. This communication between themigrating INL neuronal progenitors and the resident ONL rod precursors defines apreviously unrecognized event in regulating the regeneration response in the adult zebrafishretina. Finally, these data strongly suggest distinct mechanisms are required for selective rodand cone photoreceptor regeneration.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe thank D. Bang and the staff of the Freimann Life Science Center for providing zebrafish husbandry and care.This work was funded by National Institutes of Health grants R21EY018919 (D.R.H.) and R01EY018417 (D.R.H.and R.T.), the Center for Zebrafish Research at the University of Notre Dame, and a Fight for Sight PostdoctoralResearch fellowship (R.T.).

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radial Muller glia that function as retinal stem cells. J Neurosci 2007;27:7028–7040. [PubMed:17596452]

Bernardos RL, Raymond PA. GFAP transgenic zebrafish. Gene Expr Patterns 2006;6:1007–1013.[PubMed: 16765104]

Blader P, Lam CS, Rastegar S, Scardigli R, Nicod JC, Simplicio N, Plessy C, Fischer N, SchuurmansC, Guillemot F, Strahle U. Conserved and acquired features of neurogenin1 regulation.Development 2004;131:5627–5637. [PubMed: 15496438]

Braisted JE, Essman TF, Raymond PA. Selective regeneration of photoreceptors in goldfish retina.Development 1994;120:2409–2419. [PubMed: 7956821]

Bringmann A, Pannicke T, Grosche J, Francke M, Wiedemann P, Skatchkov SN, Osborne NN,Reichenbach A. Muller cells in the healthy and diseased retina. Prog Retin Eye Res 2006;25:397–424. [PubMed: 16839797]

Cameron DA. Cellular proliferation and neurogenesis in the injured retina of adult zebrafish. VisNeurosci 2000;17:789–797. [PubMed: 11153658]

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Fausett BV, Goldman D. A role for alpha1 tubulin-expressing Muller glia in regeneration of theinjured zebrafish retina. J Neurosci 2006;26:6303–6313. [PubMed: 16763038]

Fausett BV, Gumerson JD, Goldman D. The proneural basic helix-loop-helix gene ascl1a is requiredfor retina regeneration. J Neurosci 2008;28:1109–1117. [PubMed: 18234889]

Fimbel SM, Montgomery JE, Burket CT, Hyde DR. Regeneration of inner retinal neurons afterintravitreal injection of ouabain in zebrafish. J Neurosci 2007;27:1712–1724. [PubMed:17301179]

Hitchcock P, Kakuk-Atkins L. The basic helix-loop-helix transcription factor neuroD is expressed inthe rod lineage of the teleost retina. J Comp Neurol 2004;477:108–117. [PubMed: 15281083]

Hitchcock PF, Lindsey Myhr KJ, Easter SS Jr, Mangione-Smith R, Jones DD. Local regeneration inthe retina of the goldfish. J Neurobiol 1992;23:187–203. [PubMed: 1527527]

Hitchcock PF, Raymond PA. The teleost retina as a model for developmental and regenerationbiology. Zebrafish 2004;1:257–271. [PubMed: 18248236]

Kassen SC, Ramanan V, Montgomery JE, C TB, Liu CG, Vihtelic TS, Hyde DR. Time course analysisof gene expression during light-induced photoreceptor cell death and regeneration in albinozebrafish. Dev Neurobiol 2007;67:1009–1031. [PubMed: 17565703]

Kassen SC, Raycroft FJ, Thummel R, Plasschaert R, Hyde DR. Stat3 is required for maximal Müllerglial cell proliferation during regeneration of the light-damaged zebrafish retina. J Neurosci. 2010October; Under Review, 2009.

Lawrence JM, Singhal S, Bhatia B, Keegan DJ, Reh TA, Luthert PJ, Khaw PT, Limb GA. MIO-M1cells and similar muller glial cell lines derived from adult human retina exhibit neural stem cellcharacteristics. Stem Cells 2007;25:2033–2043. [PubMed: 17525239]

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Marcus RC, Delaney CL, Easter SS Jr. Neurogenesis in the visual system of embryonic and adultzebrafish (Danio rerio). off. Vis Neurosci 1999;16:417–424. [PubMed: 10349963]

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Figure 1. Knockdown of Pax6 in the regenerating zebrafish retinaPax6 expression (green), PCNA expression (red), and nuclei (blue) were examined duringintense light treatment. Throughout the timecourse, Pax6 expression was observed in matureamacrine cells (A, arrow in INL) and ganglion cells (A, arrow in GCL). Pax6 expression inthe photoreceptor layer, particularly in the cones (A, arrow in ONL), likely represents non-specific binding of the primary antibody as it is highly variable, even on adjacent tissuesections. Pax6 expression did not colabel with proliferating rod precursors (B, 16 hrs,arrowhead) or with proliferating Müller glia (C, 36 hrs, arrowhead). Pax6 expression wasobserved in proliferating neuronal progenitors at 72 and 96 hrs (D and E, respectively,arrows). To test the efficacy and specificity of the anti-pax6 morpholinos, zebrafish were

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either uninjected or injected and electroporated with one of two 5-base mismatch controlmorpholinos, with anti-pax6a morpholino, anti-pax6b morpholino, or a cocktail of bothmorpholinos. Pax6 expression (green) was assessed after 72 hrs of constant light treatment(Panels F–K). The uninjected (F), 5mis MO-1 (G), and 5mis MO-2 retinas (H) all containedlarge columns of neuronal progenitor cells that colabeled with Pax6 (arrows). Knockdown ofonly the Pax6a paralog (I) or both Pax6a and Pax6b (Panel K) resulted in a loss of nearly allPax6 expression. However, Pax6-positive amacrine and ganglion cells were detected in thepax6b morphant retina (J), although Pax6 expression in the columns of neuronal progenitorswas reduced. The scale bar in panel A represents 25 microns and is the same for panels B–K. GCL, ganglion cell layer; INL, inner nuclear cell layer; ONL, outer nuclear layer.

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Figure 2. Efficient and specific morpholino-induced knockdown of Pax6a and Pax6b duringembryonic developmentImmunoblot analysis with the rabbit anti-Pax6 polyclonal antisera detected both Pax6a andPax6b paralogs (51 and 47 kDa, respectively) in wild-type zebrafish embryos (lane 1). Theanti-pax6a and anti-pax6b morpholinos (pax6a MO and pax6b MO, respectively) selectivelyknockdown the expression of the expected Pax6 paralogs (lanes 2 and 3, respectively).Coinjection of both morpholinos (pax6a MO + pax6b MO) significantly reduced theexpression of both Pax6 proteins (lane 4).

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Figure 3. Pax6 is required for proliferation of Müller glial-derived neuronal progenitorsDark-adapted adult albino zebrafish were either uninjected or injected and electroporatedwith one of two 5-base mismatch control morpholinos (5mis MO-1 and 5mis MO-2), anti-pax6a morpholino, anti-pax6b morpholino, or a cocktail of both morpholinos. PCNA (red)and Glutamine synthetase (blue) expression were assessed after 72 hrs of constant lighttreatment by immunohistochemistry. The uninjected and 5mis MO retinas (A–C) allcontained large columns of PCNA-positive neuronal progenitor cells associated with Müllerglial cell processes (arrow). However, knockdown of only the Pax6a paralog (D) yieldedcolumns (arrowheads) that contained fewer PCNA-positive cells relative to controls. Incontrast, knockdown of only Pax6b (E) or both Pax6a and Pax6b (F) produced clusters ofneuronal progenitors (arrowheads) that were fewer in number than the pax6a morphantretina. The scale bar in panel A represents 25 microns and is the same for panels B–F. GCL,ganglion cell layer; INL, inner nuclear cell layer; ONL, outer nuclear layer.

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Figure 4. BrdU incorporation reveals that Pax6a and Pax6b both function at different stages ofneuronal progenitor proliferationIn uninjected retinas, after 72 hours of light treatment, BrdU (green) colabeled with PCNA(red) in columns of proliferating neuronal progenitors in the INL (A), while BrdU-positiveneuronal progenitors (green) were associated with GFAP-labeled (red) Müller glialprocesses (B, arrowhead). (C) Three days following completion of the light treatment,BrdU-positive nuclei (green) were visualized in the ONL (double arrowhead), correspondingto the daughter cells of the INL neuronal progenitors, and the individual Müller cells(arrows) that were the source of the neuronal progenitors. (D–I) After 72 hours of constantlight, BrdU-labeled nuclei (green) were observed in the ONL of control and experimental

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retinas (double arrowheads). Large clusters of BrdU-labeled neuronal progenitors were alsoobserved in the INL (arrows) of uninjected (D), the 5mis MO-1 (E), and the 5mis MO-2 (F)retinas. In contrast, pax6a morphant retinas (G) contained an average of only four BrdU-positive nuclei per cluster. Strikingly, pax6b morphant and pax6a/pax6b double morphantretinas (H and I, respectively) possessed an average of only two BrdU-labeled nuclei percluster, which was consistent with the Müller cell only dividing once and no proliferation ofthe neuronal progenitors. The scale bar in panel A represents 25 microns and is the same forpanels D–I, while the scale bar in panel B represents 25 microns and is the same for panel C.GCL, ganglion cell layer; INL, inner nuclear cell layer; ONL, outer nuclear layer.

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Figure 5. Reduced numbers of INL neuronal progenitors observed in the pax6 morphants is nota result of increased cell deathDark-adapted adult albino zebrafish were either uninjected or injected and electroporatedwith a 5-base mismatch control morpholino (5mis MO-1), anti-pax6a morpholino, or anti-pax6b morpholino. PCNA expression (red) and TUNEL labeling (blue) were assessed after72 hours of constant light treatment by immunohistochemistry. The uninjected and 5misMO-1 retinas (A, B) both contained large columns of PCNA-positive neuronal progenitorcells (arrow). However, knockdown of only Pax6a (C) yielded columns (double arrows) thatcontained fewer PCNA-positive cells relative to controls. In contrast, knockdown of onlyPax6b (D) produced clusters of neuronal progenitors (double arrowheads) that were fewer in

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number than even the pax6a morphant retina. TUNEL-positive nuclei (single arrowheads)were occasionally observed in the ONL of both the control and pax6 morphant retinas (A,C). However, no TUNEL-positive nuclei were observed in the INL of either the control orpax6 morphant retinas. The scale bar in panel A represents 25 microns and is the same forpanels B–D. GCL, ganglion cell layer; INL, inner nuclear cell layer; ONL, outer nuclearlayer.

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Figure 6. Rod precursor proliferation is independent of Pax6Dark-adapted adult albino zebrafish were separated into three control groups, eitheruninjected or injected and electroporated with one of two 5-base mismatch controlmorpholinos (A–C and G–I), and three experimental groups, injected and electroporatedwith anti-pax6a morpholino, anti-pax6b morpholino, or a cocktail of both morpholinos (D–Fand J–L), exposed to constant intense light for 96 hours, and then standard light for 3 days.Control retinas (A–C) possessed rhodopsin (green) labeling in newly regenerated rodphotoreceptors and PCNA (red) primarily in the ONL. The pax6a morphant retinasexhibited visible, but relatively reduced, expression of rhodopsin that suggested minimal rodphotoreceptor regeneration had occurred (D). Both the pax6b morphant and pax6a/pax6bdouble morphant retinas possessed only isolated rhodopsin-positive outer segments (E andF, respectively, arrows). While PCNA-positive cells in the pax6a morphant were largelyrestricted to the ONL (J), there were significantly more relative to the control retinas. Thepax6b morphant and pax6a/pax6b double morphant retinas still possessed the doublets ofPCNA-positive INL cells that were observed at 72 hours of constant light (Figs. 3 and 4). Inaddition, the pax6b morphant and pax6a/pax6b double morphant retinas contained thegreatest number of PCNA-positive cells in the ONL. The scale bar in panel A represents 25microns and is the same for panels B–L. GCL, ganglion cell layer; INL, inner nuclear celllayer; ONL, outer nuclear layer; RIS, rod inner segments; ROS, rod outer segments.

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Figure 7. Pax6 is required for cone, but not rod, cell regenerationDark-adapted adult albino zebrafish were either uninjected (A, F, K) or injected andelectroporated with the Standard Control morpholino (B, G, L), with anti-pax6a morpholino(C, H, M), anti-pax6b morpholino (D, I, N), or a cocktail of both morpholinos (E, J, O) andexposed to constant intense light for 96 hours. Retinas were examined 28 days following thecompletion of the constant light treatment. Rhodopsin immunolocalization (A–E, red)suggested that equivalent numbers of rod photoreceptors regenerated in the uninjected andmorphant retinas. The pax6b morphant and pax6a/pax6b double morphant retinas containedsignificantly fewer long and short single cones relative to the controls, as visualized by blueopsin (F–J, blue) and UV opsin (K–O, green) immunolocalization, respectively. In contrast,the pax6a morphant retinas possessed an intermediate number of long and short single conesrelative to the controls and the pax6b morphants. The scale bar in panel A represents 25microns and is the same for panels B–O.

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Figure 8. Model of zebrafish retinal regenerationA. The healthy adult zebrafish retina is made up of rod (tall light blue) and cone (short lightblue) photoreceptors that interdigitate with Müller glial cell processes (purple). B.Photoreceptors begin to undergo apoptosis (red) within six hours of beginning constantintense light treatment. The damaged photoreceptors signal (arrow) a subset of the Müllerglial cells (green) to hypertrophy and upregulate expression of Stat3 and Ascl1a. C. By 36hours, nearly all the rods and most of the cones are lost. The responding Müller glial cells(green) increase expression of the early response genes, such as stat3 and ascl1a, and reenterthe cell cycle to divide asymmetrically to produce a PCNA-positive neuronal progenitor cell(orange). D. Neuronal progenitors continue proliferating and generate the 4-cell clusteraround the Müller glia. E. Neuronal progenitor cell proliferation persists until each Müllerglial cell cluster contains an average of 8 nuclei. F. Neuronal progenitors migrate from theINL to the ONL, where they differentiate into newly regenerated rods and cones (orange),which are indistinguishable from the undamaged photoreceptors (light blue cone). ONL-resident rod precursor cells were ignored for simplicity.

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Tabl

e 1

Qua

ntifi

catio

n of

Mül

ler

glia

and

spec

ific

neur

onal

cla

sses

dur

ing

retin

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braf

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wer

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or in

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nd e

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ropo

rate

d w

ith: t

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ndar

d C

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a 5

-bas

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ism

atch

con

trol m

orph

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o (5

mis

pax

6a M

O a

nd 5

mis

pax

6b M

O),

anti-

pax6

a m

orph

olin

o, a

nti-p

ax6b

mor

phol

ino,

or a

coc

ktai

l of b

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anti-

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phol

inos

. Exp

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of M

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and

neu

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l-spe

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cel

l mar

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e as

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us ti

mes

of c

onst

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ight

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chem

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. Qua

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was

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a 7

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OV

A a

naly

sis (

p) a

re sh

own

for e

ach

expe

rimen

t.St

anda

rd d

evia

tions

are

show

n in

par

enth

eses

and

ast

eris

ks re

pres

ent d

ata

is si

gnifi

cant

ly d

iffer

ent f

rom

the

cont

rols

usi

ng B

onfe

rron

i-cor

rect

ed t-

test

s.

Ave

rage

per

cent

age

of g

luta

min

e sy

nthe

tase

-pos

itive

Mül

ler g

lia th

at a

re P

CN

A-p

ositi

ve

Tim

e po

int

Np

Uni

njec

ted

Std.

Ctl.

MO

5mis

pax

6a M

O5m

is p

ax6b

MO

pax6

a M

Opa

x6b

MO

pax6

a/6b

MO

36 h

rs li

ght t

xt6

0.79

254

.6 (2

.8)

48.1

(5.8

)N

.A.

N.A

.51

.3 (2

.5)

53.2

(4.5

)49

.8 (3

.7)

Ave

rage

num

ber o

f PC

NA

-pos

itive

nuc

lei p

er M

ülle

r glia

l clu

ster

Tim

e po

int

Np

Uni

njec

ted

Std.

Ctl.

MO

5mis

pax

6a M

O5m

is p

ax6b

MO

pax6

a M

Opa

x6b

MO

pax6

a/6b

MO

72 h

rs li

ght t

xt6

<0.0

017.

9 (0

.7)

7.3

(0.5

)7.

7 (0

.1)

8.1

(0.3

)3.

8 (0

.1) *

1.9

(0.2

) *2.

1 (0

.2) *

Ave

rage

num

ber o

f Brd

U-p

ositi

ve n

ucle

i per

Mül

ler g

lial c

lust

er

Tim

e po

int

Np

Uni

njec

ted

Std.

Ctl.

MO

5mis

pax

6a M

O5m

is p

ax6b

MO

pax6

a M

Opa

x6b

MO

pax6

a/6b

MO

72 h

rs li

ght t

xt10

<0.0

017.

4 (0

.6)

N.A

.8.

0 (0

.1)

8.1

(0.1

)4.

6 (0

.1) *

2.3

(0.1

) *2.

0 (0

.1) *

Ave

rage

num

ber o

f PC

NA

-pos

itive

nuc

lei i

n O

NL

Tim

e po

int

Np

Uni

njec

ted

Std.

Ctl.

MO

5mis

pax

6a M

O5m

is p

ax6b

MO

pax6

a M

Opa

x6b

MO

pax6

a/6b

MO

3 da

ys p

ost t

xt6

<0.0

0176

.2 (4

.5)

75.8

(5.6

)78

.4 (5

.0)

70.5

(5.6

)11

8.4

(20.

3)16

1.0

(11.

4) *

158.

6 (1

1.7)

*

Ave

rage

num

ber o

f Mül

ler g

lial c

ells

Tim

e po

int

Np

Uni

njec

ted

Std.

Ctl.

MO

5mis

pax

6a M

O5m

is p

ax6b

MO

pax6

a M

Opa

x6b

MO

pax6

a/6b

MO

28 d

ays p

ost t

xt8

0.82

770

.6 (2

.6)

69.1

(4.0

)N

.A.

N.A

.71

.4 (1

.1)

68.2

(2.7

)69

.0 (1

.8)

Ave

rage

num

ber o

f ON

L ro

d ph

otor

ecep

tor n

ucle

i

Tim

e po

int

Np

Uni

njec

ted

Std.

Ctl.

MO

5mis

pax

6a M

O5m

is p

ax6b

MO

pax6

a M

Opa

x6b

MO

pax6

a/6b

MO

28 d

ays p

ost t

xt8

0.84

531

5.8

(12.

0)30

7.6

(12.

4)33

5.4

(12.

7)30

8.5

(11.

0)31

8.4

(16.

3)32

2.2

(17.

5)31

3.6

(16.

4)

Ave

rage

num

ber o

f lon

g si

ngle

con

e ce

lls


NIH


NIH


NIH


Thummel et al.

Tim

e po

int

Np

Uni

njec

ted

Std.

Ctl.

MO

5mis

pax

6a M

O5m

is p

ax6b

MO

pax6

a M

Opa

x6b

MO

pax6

a/6b

MO

28 d

ays p

ost t

xt8

<0.0

0149

.2 (5

.1)

48.2

(4.5

)50

.2 (4

.8)

48.4

(5.5

)34

.8 (2

.3)

20.8

(2.8

) *19

.0 (1

.9) *


Pax6a and Pax6b are required at different points in neuronal progenitor cell proliferation during...

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